title: The Phosphate Source Influences Gene Expression and Quality of Mineralization during In Vitro Osteogenic Differentiation of Human Mesenchymal Stem Cells.
abstract: For in vitro differentiation of bone marrow-derived mesenchymal stem cells/mesenchymal stromal cells into osteoblasts by 2-dimensional cell culture a variety of protocols have been used and evaluated in the past. Especially the external phosphate source used to induce mineralization varies considerably both in respect to chemical composition and concentration. In light of the recent findings that inorganic phosphate directs gene expression of genes crucial for bone development, the need for a standardized phosphate source in in vitro differentiation becomes apparent. We show that chemical composition (inorganic versus organic phosphate origin) and concentration of phosphate supplementation exert a severe impact on the results of gene expression for the genes commonly used as markers for osteoblast formation as well as on the composition of the mineral formed. Specifically, the intensity of gene expression does not necessarily correlate with a high quality mineralized matrix. Our study demonstrates advantages of using inorganic phosphate instead of β-glycerophosphate and propose colorimetric quantification methods for calcium and phosphate ions as cost- and time-effective alternatives to X-ray diffraction and Fourier-transform infrared spectroscopy for determination of the calcium phosphate ratio and concentration of mineral matrix formed under in vitro-conditions. We critically discuss the different assays used to assess in vitro bone formation in respect to specificity and provide a detailed in vitro protocol that could help to avoid contradictory results due to variances in experimental design.
authors.affiliation: Trauma Department, Medical School Hannover, Hannover, Germany.
authors.forename: Luisa M
authors.initials: LM
authors.lastname: Schäck
authors.forename: Sandra
authors.initials: S
authors.lastname: Noack
authors.forename: Ramona
authors.initials: R
authors.lastname: Winkler
authors.forename: Gesa
authors.initials: G
authors.lastname: Wißmann
authors.forename: Peter
authors.initials: P
authors.lastname: Behrens
authors.forename: Mathias
authors.initials: M
authors.lastname: Wellmann
authors.forename: Michael
authors.initials: M
authors.lastname: Jagodzinski
authors.forename: Christian
authors.initials: C
authors.lastname: Krettek
authors.forename: Andrea
authors.initials: A
authors.lastname: Hoffmann
mesh_terms: D015415:Biomarkers; D002113:Calcification, Physiologic; D002454:Cell Differentiation; D015870:Gene Expression; D006801:Humans; D066298:In Vitro Techniques; D059630:Mesenchymal Stem Cells / Q000166:cytology*; D010012:Osteogenesis; D010710:Phosphates / Q000378:metabolism*; D017550:Spectroscopy, Fourier Transform Infrared; D014961:X-Ray Diffraction
chemical_list: D015415:Biomarkers; D010710:Phosphates